Expansion of undifferentiated pluripotent stem cells has been traditionally employed “feeder” cells which provide sufficient factors to support attachment, proliferation and maintenance of pluripotency markers. Early methods for the generation and culture of human embryonic stem cells required the use of mouse embryonic fibroblast (MEF) feeder cells. Subsequent techniques included use of “conditioned media” and an extracellular matrix coating to replace feeder cells. Conditioned media is media that has been modified by feeder cells, such as MEFs. However, both methods suffer from inconsistencies in batches of conditioned media or feeder cells to continually support expansion of pluripotent stem cells. Furthermore, both systems provide undefined factors that may work differently on different pluripotent stem cells. Accordingly, establishing a defined, cheap, reproducible culture media that supports continual expansion of pluripotent stem cells is of great interest in the regenerative medicine field.
A defining feature of human embryonic stem cells (hES cells) is that the cells have a tendency to differentiate into various lineages. This unwanted differentiation can hamper uniform and directed differentiation required to subsequently generate desired specific cell types. In fact, both feeder cells and conditioned media culture conditions typically result in some level of unwanted differentiation, particularly around the edges of the growing ES cell colony or in the center of the colony.
Recent efforts have resulted in replacement of feeder cells or conditioned media with a host of replacement culture conditions, such as knock-out serum replacer (KSR) in the media (Chen et al., 2005, Nature Methods, 2:185-189). KSR contains a crude undefined fraction of bovine serum albumin (BSA). Others have shown long-term maintenance of pluripotency in a chemically defined media with FGF2, activin A, and insulin (Vallier et al., 2005, J Cell Sci, 118:4495-4509) Commercially available media formulations including mTeSR®1 media (StemCell Technologies, Vancouver, Canada) and StemPro™ (Invitrogen, CA) have also been previously used to maintain and proliferate human pluripotent stem cells. Additional prior art focusing on development of defined media include U.S. Pat. No. 7,449,334, U.S. Pat. No. 7,442,548, U.S. Pat. No. 7,005,252, US2008/0268534, U.S. Pat. No. 7,410,798, U.S. Pat. No. 7,297,539, and U.S. Pat. No. 6,800,480. Furthermore, a recent publication further refined the mTeSR®1 media to eight components (Nature Methods, 2011, 8:424-429) highlighting that even in defined media there exists unnecessary agent(s) that may actually slow the proliferation of ES cells or reduce their pluripotency state. The refined mTeSR®1 media consists of DMEM/F12 basal media supplemented with insulin, selenium, transferrin, ascorbic acid, FGF2 (bFGF), and TGFβ or nodal, having the pH adjusted with NaHCO3.
It is therefore clear that there is still a need for fully defined media conditions that provide consistency regarding expansion of pluripotent cells while having minimal number of added components.